scholarly journals Triclosan depletes the membrane potential in Pseudomonas aeruginosa biofilms inhibiting aminoglycoside induced adaptive resistance

2020 ◽  
Author(s):  
Michael M. Maiden ◽  
Christopher M. Waters
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Potential ◽  
Efflux Pump ◽  
Antibacterial Properties ◽  
Efflux Pumps ◽  
Chronic Infections ◽  
Onset Of Action ◽  
Adaptive Resistance

AbstractBiofilm-based infections are difficult to treat due to their inherent resistance to antibiotic treatment. Discovering new approaches to enhance antibiotic efficacy in biofilms would be highly significant in treating many chronic infections. Exposure to aminoglycosides induces adaptive resistance in Pseudomonas aeruginosa biofilms. Adaptive resistance is primarily the result of active antibiotic export by RND-type efflux pumps, which use the proton motive force as an energy source. We show that the protonophore uncoupler triclosan depletes the membrane potential of biofilm growing P. aeruginosa, leading to decreased activity of RND-type efflux pumps. This disruption results in increased intracellular accumulation of tobramycin and enhanced antimicrobial activity in vitro. In addition, we show that triclosan enhances tobramycin effectiveness in vivo using a mouse wound model. Combining triclosan with tobramycin is a new anti-biofilm strategy that targets bacterial energetics, increasing the susceptibility of P. aeruginosa biofilms to aminoglycosides.Author summaryAdaptive resistance is a phenotypic response that allows P. aeruginosa to transiently survive aminoglycosides such as tobramycin. To date, few compounds have been identified that target adaptive resistance. Here, we show the protonophore uncoupler triclosan disrupts the membrane potential of P. aeruginosa. The depletion of the membrane potential reduces efflux pump activity, which is essential for adaptive resistance, leading to increased tobramycin accumulation and a shorter onset of action. Our results demonstrate that in addition to its canonical mechanism inhibiting membrane biosynthesis, triclosan can exert antibacterial properties by functioning as a protonophore that targets P. aeruginosa energetics.

scholarly journals Triclosan depletes the membrane potential in Pseudomonas aeruginosa biofilms inhibiting aminoglycoside induced adaptive resistance

2020 ◽  
Vol 16 (10) ◽  
pp. e1008529
Author(s):  
Michael M. Maiden ◽  
Christopher M. Waters
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Potential ◽  
Efflux Pumps ◽  
Chronic Infections ◽  
Adaptive Resistance ◽  
Wound Model ◽  
Model Combining ◽  
Antibiotic Efficacy

Biofilm-based infections are difficult to treat due to their inherent resistance to antibiotic treatment. Discovering new approaches to enhance antibiotic efficacy in biofilms would be highly significant in treating many chronic infections. Exposure to aminoglycosides induces adaptive resistance in Pseudomonas aeruginosa biofilms. Adaptive resistance is primarily the result of active antibiotic export by RND-type efflux pumps, which use the proton motive force as an energy source. We show that the protonophore uncoupler triclosan depletes the membrane potential of biofilm growing P. aeruginosa, leading to decreased activity of RND-type efflux pumps. This disruption results in increased intracellular accumulation of tobramycin and enhanced antimicrobial activity in vitro. In addition, we show that triclosan enhances tobramycin effectiveness in vivo using a mouse wound model. Combining triclosan with tobramycin is a new anti-biofilm strategy that targets bacterial energetics, increasing the susceptibility of P. aeruginosa biofilms to aminoglycosides.

scholarly journals Pharmacodynamics of Levofloxacin against Pseudomonas aeruginosa with Reduced Susceptibility Due to Different Efflux Pumps: Do Elevated MICs Always Predict Reduced In Vivo Efficacy?

2006 ◽  
Vol 50 (5) ◽  
pp. 1628-1632 ◽  
Author(s):  
David C. Griffith ◽  
Erik Corcoran ◽  
Denene Lofland ◽  
Angela Lee ◽  
Deidre Cho ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Pharmacodynamic Modeling ◽  
Time Curve ◽  
Effective Doses ◽  
Isogenic Strains ◽  
Proportional Reduction

ABSTRACT The Pseudomonas aeruginosa efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN play an important role in susceptibility to fluoroquinolones in vitro. To determine if levofloxacin MICs arising from different levels of expression of efflux pumps result in a proportional reduction in the response to levofloxacin in vivo, isogenic strains of P. aeruginosa were tested with levofloxacin in two mouse models of infection (sepsis and neutropenic mouse thigh models). The levofloxacin 50% effective doses (ED50s) increased proportionally with the MICs for most strains. Similarly, the 24-h area under the concentration-time curve (AUC)/MIC ratio that resulted in 90% of the maximum bactericidal activity (90% E max) exceeded 75 for all strains except those with elevated MICs due to MexEF-OprN overexpression. In these strains, levofloxacin ED50s were 2- to 10-fold lower than the ED50/MIC ratios in the other strains and 90% E max AUC/MIC ratios were 2- to 4-fold lower than those predicted from pharmacodynamic modeling of efficacy against other strains. These data show that while the MexEF-OprN efflux pump can provide P. aeruginosa resistance to levofloxacin in vitro, it appears to be less efficient in providing resistance to levofloxacin in animal models of infection.

scholarly journals Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model.

1997 ◽  
Vol 41 (4) ◽  
pp. 823-826 ◽  
Author(s):  
Y Q Xiong ◽  
J Caillon ◽  
M F Kergueris ◽  
H Drugeon ◽  
D Baron ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Single Dose ◽  
Ex Vivo ◽  
Day Treatment ◽  
Total Daily Dose ◽  
Once Daily ◽  
Adaptive Resistance ◽  
Dosing Regimens

Adaptive resistance following the first exposure to aminoglycosides is a recently described in vitro phenomenon in Pseudomonas aeruginosa and other aerobic gram-negative bacilli. We investigated the in vivo relevance of adaptive resistance in P. aeruginosa following a single dose of amikacin in the experimental rabbit endocarditis model. Rabbits with P. aeruginosa endocarditis received either no therapy (control) or a single intravenous (i.v.) dose of amikacin (80 mg/kg of body weight) at 24 h postinfection, after which they were sacrificed at 5, 8, 12, 16, or 24 h postdose. Excised aortic vegetations were subsequently exposed ex vivo to amikacin at 2.5, 5, 10 or 20 times the MIC for 90 min. In vivo adaptive resistance was identified when amikacin-induced pseudomonal killing within excised aortic vegetations was less in animals receiving single-dose amikacin in vivo than in vegetations from control animals not receiving amikacin in vivo. Maximal adaptive resistance occurred between 8 and 16 h after the in vivo amikacin dose, with complete refractoriness to ex vivo killing by amikacin seen at 12 h postdose. By 24 h postdose, bacteria within excised vegetations had partially recovered their initial amikacin susceptibility. In a parallel treatment study, we demonstrated that amikacin given once daily (but not twice daily) at a total dose of 80 mg/kg i.v. for 1-day treatment significantly reduced pseudomonal densities within aortic vegetations versus those in untreated controls. When therapy was continued for 3 days with the same total daily dose (80 mg/kg/day), amikacin given once or twice daily significantly reduced intravegetation pseudomonal densities versus those in controls. However, amikacin given once daily was still more effective than the twice-daily regimen. These data confirm the induction of aminoglycoside adaptive resistance in vivo and further support the advantages of once-daily aminoglycoside dosing regimens in the treatment of serious pseudomonal infections.

scholarly journals Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

2012 ◽  
Vol 56 (9) ◽  
pp. 4771-4778 ◽  
Author(s):  
Bartolomé Moyá ◽  
Alejandro Beceiro ◽  
Gabriel Cabot ◽  
Carlos Juan ◽  
Laura Zamorano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Binding Protein ◽  
Efflux Pumps ◽  
Binding Affinities ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Reverse Transcription Pcr

ABSTRACTWe investigated the mechanisms leading toPseudomonas aeruginosapan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence ofoprD,ampD, anddacBmutations was explored by PCR followed by sequencing and the expression ofampCand efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressedampCand one or several efflux pumps, particularlymexBand/ormexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n= 1) or diminished expression (n= 1) of PBP1a and a lack of PBP4 expression (n= 4), which correlated with AmpC overexpression driven bydacBmutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in thein vivoemergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.

scholarly journals Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

2012 ◽  
Vol 56 (4) ◽  
pp. 2084-2090 ◽  
Author(s):  
Astrid Pérez ◽  
Margarita Poza ◽  
Ana Fernández ◽  
Maria del Carmen Fernández ◽  
Susana Mallo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

scholarly journals Characterization of a mucoid-like Pseudomonas aeruginosa biofilm

Fine Focus ◽  
2015 ◽  
Vol 1 (2) ◽  
pp. 121-137
Author(s):  
Brandon M. Bauer ◽  
Lewis Rogers ◽  
Monique Macias ◽  
Gabriella Iacovetti ◽  
Alexander M. Woodrow ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Growth Conditions ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chronic Infections ◽  
Planktonic Bacteria ◽  
The Difference

Pseudomonas aeruginosa biofilms are implicated in chronic infections. A key element of P. aeruginosapathogenicity is the formation of a biofilm, a community of bacteria encased in an exopolymeric substance (EPS) that shields the bacteria from the host immune response and antibiotic treatment. A crucial step in biofilm production is a switch in motility from freely swimming, planktonic bacteria to twitching movement and then to attached and sedentary bacteria that develop into a mature pillar-shaped biofilm. A mucoid biofilm produces an excess of alginate and is clinically the most pathogenic and the most resistant to antibiotics. Biofilms from patients exhibit a wide variety of structure, motility, and levels of attachment. In vitrobiofilms do not exhibit such a wide variety of structure and physiology. The difference between in vivo and in vitro biofilms has made the translation of in vitro studies into in vivo treatments difficult. Under different growth conditions in our lab, the P. aeruginosa strain PAO1 demonstrates two phenotypes: a non-mucoid and a mucoid-like phenotype. Confocal laser scanning microscopy (CLSM) indicates the mucoid-like phenotype is intermediate in height to the non-mucoid phenotype and biofilms formed in a once-flow-through chamber. Both mucoid-like and non-mucoid phenotypes exhibit a significant increase in twitching between 24 and 72 hours of development. The mucoid-like phenotype had greater attachment at 72 hours compared to non-mucoid phenotype. Therefore, the two phenotypes observed in our lab may represent the effect of environment to stimulate development of two types of biofilms by PAO1.

scholarly journals Pseudomonas aeruginosa Forms Biofilms in Acute Infection Independent of Cell-to-Cell Signaling

2007 ◽  
Vol 75 (8) ◽  
pp. 3715-3721 ◽  
Author(s):  
J. Andy Schaber ◽  
W. Jeffrey Triffo ◽  
Sang Jin Suh ◽  
Jeffrey W. Oliver ◽  
Mary Catherine Hastert ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Colonization ◽  
Acute Infection ◽  
Opportunistic Pathogen ◽  
Confocal Scanning Laser Microscopy ◽  
Chronic Infections ◽  
Signaling Mechanism ◽  
Confocal Scanning Laser

ABSTRACT Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

scholarly journals NorB, an Efflux Pump in Staphylococcus aureus Strain MW2, Contributes to Bacterial Fitness in Abscesses

2008 ◽  
Vol 190 (21) ◽  
pp. 7123-7129 ◽  
Author(s):  
Yanpeng Ding ◽  
Yoshikuni Onodera ◽  
Jean C. Lee ◽  
David C. Hooper
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Single Copy ◽  
Aureus Strain ◽  
Bacterial Survival ◽  
Bacterial Fitness

ABSTRACT While remaining a major problem in hospitals, Staphylococcus aureus is now spreading in communities. Strain MW2 (USA400 lineage) and other community methicillin-resistant S. aureus strains most commonly cause skin infections with abscess formation. Multidrug resistance (MDR) efflux pumps contribute to antimicrobial resistance but may also contribute to bacterial survival by removal of environmental toxins. In S. aureus, NorA, NorB, NorC, and Tet38 are chromosomally encoded efflux pumps whose overexpression can confer MDR to quinolones and other compounds (Nor pumps) or tetracyclines alone (Tet38), but the natural substrates of these pumps are not known. To determine the role of these efflux pumps in a natural environment in the absence of antibiotics, we used strain MW2 in a mouse subcutaneous abscess model and compared pump gene expression as determined by reverse transcription-PCR in the abscesses and in vitro. norB and tet38 were selectively upregulated in vivo more than 171- and 24-fold, respectively, whereas norA and norC were downregulated. These changes were associated with an increase in expression of mgrA, which encodes a transcriptional regulator known to affect pump gene expression. In competition experiments using equal inocula of a norB or tet38 mutant and parent strain MW2, each mutant exhibited growth defects of about two- to threefold in vivo. In complementation experiments, a single-copy insertion of norB (but not a single-copy insertion of tet38) in the attB site within geh restored the growth fitness of the norB mutant in vivo. Our findings indicate that some MDR pumps, like NorB, can facilitate bacterial survival when they are overexpressed in a staphylococcal abscess and may contribute to the relative resistance of abscesses to antimicrobial therapy, thus linking bacterial fitness and resistance in vivo.

scholarly journals Specific and Global RNA Regulators in Pseudomonas aeruginosa

2021 ◽  
Vol 22 (16) ◽  
pp. 8632
Author(s):  
Petra Pusic ◽  
Elisabeth Sonnleitner ◽  
Udo Bläsi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Binding ◽  
Gene Networks ◽  
Target Genes ◽  
Opportunistic Pathogen ◽  
Chronic Infections ◽  
Intrinsic Resistance ◽  
Large Gene

Pseudomonas aeruginosa (Pae) is an opportunistic pathogen showing a high intrinsic resistance to a wide variety of antibiotics. It causes nosocomial infections that are particularly detrimental to immunocompromised individuals and to patients suffering from cystic fibrosis. We provide a snapshot on regulatory RNAs of Pae that impact on metabolism, pathogenicity and antibiotic susceptibility. Different experimental approaches such as in silico predictions, co-purification with the RNA chaperone Hfq as well as high-throughput RNA sequencing identified several hundreds of regulatory RNA candidates in Pae. Notwithstanding, using in vitro and in vivo assays, the function of only a few has been revealed. Here, we focus on well-characterized small base-pairing RNAs, regulating specific target genes as well as on larger protein-binding RNAs that sequester and thereby modulate the activity of translational repressors. As the latter impact large gene networks governing metabolism, acute or chronic infections, these protein-binding RNAs in conjunction with their cognate proteins are regarded as global post-transcriptional regulators.

scholarly journals Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy

2015 ◽  
Vol 59 (12) ◽  
pp. 7743-7752 ◽  
Author(s):  
Aryun Kim ◽  
Amy Kutschke ◽  
David E. Ehmann ◽  
Sara A. Patey ◽  
Jared L. Crandon ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Body Weight ◽  
Iron Uptake ◽  
Threshold Concentration ◽  
Content Type ◽  
Adaptive Resistance ◽  
Mueller Hinton ◽  
Mueller Hinton Broth

ABSTRACTThe objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 inPseudomonas aeruginosaby using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of thein vivomodel, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake:piuA,piuC,pirR,fecI, andpvdS. Against fourP. aeruginosaisolates, SMC-3176 displayed predictable efficacy corresponding to thefT>MIC using the MIC in CDMHB (R2= 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate forP. aeruginosaisolate JJ 4-36, as thein vivoresponses were inconsistent withfT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against whichin vivofailures of another siderophore-conjugated β-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy inP. aeruginosadue to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantialin vivotesting is warranted for compounds using the siderophore approach to thoroughly screen for thisin vitro-in vivodisconnect inP. aeruginosa.

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